Chemical exposure of cells may damage biomolecules, cellular structures, and organelles thereby jeopardizing cellular homeostasis. A multitude of defense mechanisms have evolved that can recognize specific types of damaged molecules and will initiate distinct cellular programs aiming to remove the damage inflicted and prevent cellular havoc. As a consequence, quantitative assessment of the activity of the cellular stress responses may serve as a sensitive reporter for the induction of specific types of damage. We have previously developed the ToxTracker assay, a mammalian stem cell-based genotoxicity assay employing two green fluorescent protein reporters specific for DNA damage and oxidative stress. We have now expanded the ToxTracker assay with an additional four reporter cell lines to include monitoring of additional stress signaling pathways. This panel of six green fluorescent protein reporters is able to discriminate between different primary reactivity of chemicals being their ability to react with DNA and block DNA replication, induce oxidative stress, activate the unfolded protein response, or cause a general P53-dependent cellular stress response. Extensive validation using the compound library suggested by the European Centre for the Validation of Alternative Methods (ECVAM) and a large panel of reference chemicals shows that the ToxTracker assay has an outstanding sensitivity and specificity. In addition, we developed Toxplot, a dedicated software tool for automated data analysis and graphical representation of the test results. Rapid and reliable identification by the ToxTracker assay of specific biological reactivity can significantly improve in vitro human hazard assessment of chemicals.

Nickel (Ni) compounds are classified as carcinogenic to humans but the underlying mechanisms are still poorly understood. Furthermore, effects related to nanoparticles (NPs) of Ni have not been fully elucidated. The aim of this study was to investigate genotoxicity and mutagenicity of Ni and NiO NPs and compare the effect to soluble Ni from NiCl2. We employed different models; i.e., exposure of (1) human bronchial epithelial cells (HBEC) followed by DNA strand break analysis (comet assay and γ‐H2AX staining); (2) six different mouse embryonic stem (mES) reporter cell lines (ToxTracker) that are constructed to exhibit fluorescence upon the induction of various pathways of relevance for (geno)toxicity and cancer; and (3) mES cells followed by mutagenicity testing (Hprt assay). The results showed increased DNA strand breaks (comet assay) for the NiO NPs and at higher doses also for the Ni NPs whereas no effects were observed for Ni ions/complexes from NiCl2. By employing the reporter cell lines, oxidative stress was observed as the main toxic mechanism and protein unfolding occurred at cytotoxic doses for all three Ni‐containing materials. Oxidative stress was also detected in the HBEC cells following NP‐exposure. None of these materials induced the reporter related to direct DNA damage and stalled replication forks. A small but statistically significant increase in Hprt mutations was observed for NiO but only at one dose. We conclude that Ni and NiO NPs show more pronounced (geno)toxic effects compared to Ni ions/complexes, indicating more serious health concerns. Environ. Mol. Mutagen. 59:211–222, 2018. © 2017 The Authors Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society

BackgroundBreast cancer is a heterogeneous disease with a highly variable clinical outcome in which both genetic and epigenetic changes have critical roles. We investigated tumor expression levels of histone-modifying enzymes LSD1, HDAC2 and SIRT1 in relation with patient survival and tumor relapse in a retrospective cohort of 460 breast cancer patients. Additionally, we correlated expression levels with tumor differentiation and tumor cell proliferation.MethodsImmunohistochemical staining for LSD1, HDAC2 and SIRT1 was performed on tissue microarrays of tumor and corresponding normal formalin-fixed paraffin-embedded tissues from breast cancer patients. Median nuclear expression levels in tumor tissues were used to divide the patients into low and high expression categories. In combined expression analyses, patients were divided into four subgroups: 1, all enzymes below-median; 2, one enzyme above-median; 3, two enzymes above-median; 4, all three enzymes above-median. The Cox proportional hazard model was used for univariate and multivariate survival analyses. The Pearson Chi-square method was used to assess correlation of combined expression levels with tumor cell proliferation and tumor differentiation.ResultsExpression of LSD1 and SIRT1, but not of HDAC2, was significantly increased in tumor tissues compared to their normal counterparts (both p < 0.001). Multivariate survival analyses identified SIRT1 as independent prognostic factor for relapse-free survival (RFS) with a hazard ratio (HR) of 1.34 (95% CI = 1.04-1.74, p = 0.02). For overall survival (OS), no significant differences were found when the individual enzymes were analyzed. Analyses of combined expression levels of the three histone-modifying enzymes correlated with OS (p = 0.03) and RFS (p = 0.006) with a HR of respectively 1.49 (95% CI = 1.07-2.08) and 1.68 (95% CI = 1.16-2.44) in multivariate analyses and were also related to tumor differentiation (p < 0.001) and tumor cell proliferation (p = 0.002).ConclusionsWhen the combined expression levels were analyzed, high expression of LSD1, HDAC2 and SIRT1 showed shorter patient survival time and shorter time to tumor relapse and correlated with poor tumor differentiation and a high level of tumor cell proliferation. Expression of these histone-modifying enzymes might therefore be involved in breast cancer pathogenesis.

An increasing use of cobalt (Co)-based nanoparticles (NPs) in different applications and exposures at occupational settings triggers the need for toxicity assessment. Improved understanding regarding the physiochemical characteristics of Co metal NPs and different oxides in combination with assessment of toxicity and mechanisms may facilitate decisions for grouping during risk assessment. The aim of this study was to gain mechanistic insights in the correlation between NP reactivity and toxicity of three different Co-based NPs (Co, CoO, and CoO) by using various tools for characterization, traditional toxicity assays, as well as six reporter cell lines (ToxTracker) for rapid detection of signaling pathways of relevance for carcinogenicity. The results showed cellular uptake of all NPs in lung cells and induction of DNA strand breaks and oxidative damage (comet assay) by Co and CoO NPs. In-depth studies on the ROS generation showed high reactivity of Co, lower for CoO, and no reactivity of CoO NPs. The reactivity depended on the corrosion and transformation/dissolution properties of the particles and the media highlighting the role of the surface oxide and metal speciation as also confirmed by in silico modeling. By using ToxTracker, Co NPs were shown to be highly cytotoxic and induced reporters related to oxidative stress (Nrf2 signaling) and DNA strand breaks. Similar effects were observed for CoO NPs but at higher concentrations, whereas the CoO NPs were inactive at all concentrations tested. In conclusion, our study suggests that Co and CoO NPs, but not CoO, may be grouped together for risk assessment.

Millions of people in the world perform welding as their primary occupation resulting in exposure to metal-containing nanoparticles in the fumes generated. Even though health effects including airway diseases are well-known, there is currently a lack of studies investigating how different welding setups and conditions affect the toxicity of generated nanoparticles of the welding fume. The aim of this study was to investigate the toxicity of nine types of welding fume particles generated via active gas shielded metal arc welding (GMAW) of chromiumcontaining stainless steel under different conditions and, furthermore, to correlate the toxicity to the particle characteristics. Toxicological endpoints investigated were generation of reactive oxygen species (ROS), cytotoxicity, genotoxicity and activation of ToxTracker reporter cell lines. The results clearly underline that the choice of filler material has a large influence on the toxic potential. Fume particles generated by welding with the tested flux-cored wire (FCW) were found to be more cytotoxic compared to particles generated by welding with solid wire or metal-cored wire (MCW). FCW fume particles were also the most potent in causing ROS and DNA damage and they furthermore activated reporters related to DNA double-strand breaks and p53 signaling. Interestingly, the FCW fume particles were the most soluble in PBS, releasing more chromium in the hexavalent form and manganese compared to the other fumes. These results emphasize the importance of solubility of different metal constituents of the fume particles, rather than the total metal content, for their acute toxic potential.

Background: Colon cancer prognosis and treatment are currently based on a classification system still showing large heterogeneity in clinical outcome, especially in TNM stages II and III. Prognostic biomarkers for metastasis risk are warranted as development of distant recurrent disease mainly accounts for the high lethality rates of colon cancer. miRNAs have been proposed as potential biomarkers for cancer. Furthermore, a verified standard for normalization of the amount of input material in PCR-based relative quantification of miRNA expression is lacking.Methods: A selection of frozen tumor specimens from two independent patient cohorts with TNM stage II-III microsatellite stable primary adenocarcinomas was used for laser capture microdissection. Next-generation sequencing was performed on small RNAs isolated from colorectal tumors from the Dutch cohort (N ¼ 50). Differential expression analysis, comparing in metastasized and nonmetastasized tumors, identified prognostic miRNAs.

Multiple studies showed the prognostic capacities of tumor-infiltrating lymphocytes (TILs) in triple-negative breast cancer (TNBC), but not in other subtypes. We evaluated tumor expression of FAS, a key receptor in T-cell mediated apoptosis, as possible explanation for this differential prognostic value of TILs. Furthermore, we evaluated the prognostic relevance of FAS, both as an independent biomarker and in relation to CD8-positive T-cell presence. The study cohort consisted of 667 breast cancer patients treated in the LUMC between 1997 and 2009. FAS expression was determined using immunohistochemistry and the percentage of FAS-positive tumor cells was quantified. Furthermore, the number of CD8-positive infiltrating cells was determined, and its prognostic relevance was associated to FAS-expression using stratified survival analysis. In TNBC, FAS was averagely expressed in 49% of tumor cells, whereas ER-positive subtypes showed an average Fas expression of 16-20%. In the entire cohort, FAS was identified as significant prognostic marker for recurrence (adjusted HR 0.53, 95% CI 0.36-0.77) and borderline significant marker for overall survival (adjusted HR 0.72, 95% CI 0.52-1.01). Upon stratification for FAS-expression, CD8+ TILs were only prognostic at high levels (above median) of FAS expression in ER-negative disease. In summary, FAS was identified as an independent prognostic marker for recurrence free survival in breast cancer, with large variation in expression by receptor subtypes. Interestingly, the prognostic effect of CD8+ TILs in ER-negative disease was only valid for tumors with a high FAS expression.

The increased use of nanoparticles (NPs) requires efficient testing of their potential toxic effects. A promising approach is to use reporter cell lines to quickly assess the activation of cellular stress response pathways. This study aimed to use the ToxTracker reporter cell lines to investigate (geno)toxicity of various metal- or metal oxide NPs and draw general conclusions on NP-induced effects, in combination with our previous findings. The NPs tested in this study (n = 18) also included quantum dots (QDs) in different sizes. The results showed a large variation in cytotoxicity of the NPs tested. Furthermore, whereas many induced oxidative stress only few activated reporters related to DNA damage. NPs of manganese (Mn and Mn3O4) induced the most remarkable ToxTracker response with activation of reporters for oxidative stress, DNA damage, protein unfolding and p53-related stress. The QDs (CdTe) were highly toxic showing clearly size-dependent effects and calculations suggest surface area as the most relevant dose metric. Of all NPs investigated in this and previous studies the following induce the DNA damage reporter; CuO, Co, CoO, CdTe QDs, Mn, Mn3O4, V2O5, and welding NPs. We suggest that these NPs are of particular concern when considering genotoxicity induced by metal- and metal oxide NPs.